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Abstract

Background—The AT1 receptor has been implicated in the pathogenesis of hypertension and atherosclerosis. Estrogen deficiency is also associated with cardiovascular diseases. Therefore, we examined the AT1 receptor gene expression in ovariectomized rats with and without estrogen replacement therapy and the influence of estrogen on AT1 receptor expression in cultured vascular smooth muscle cells.

Methods and Results—Rat aortic tissue was examined 5 weeks after ovariectomy. In one group, estrogen (1.7 mg estradiol) was administered during the 5-week period. Functional experiments assessed angiotensin II–induced contraction of aortic rings. AT1 receptor mRNA levels were measured by quantitative polymerase chain reaction and Northern blotting. AT1 receptor density was assessed by radioligand binding assays. These techniques were also applied in cultured vascular smooth muscle cells. The efficacy of angiotensin II on vasoconstriction was significantly increased in aortas from ovariectomized rats. As assessed by radioligand binding assays, AT1 receptor density was increased to 160% without changes in receptor affinity during estrogen deficiency. AT1 receptor mRNA levels were consistently increased to 187% in ovariectomized rats compared with sham-operated animals. Estrogen substitution therapy in ovariectomized rats reversed this AT1 receptor overexpression. To explore the underlying mechanisms, the direct influence of estradiol on AT1 receptor expression was investigated in VSMCs. Estradiol (1 μmol/L) led to a time-dependent downregulation of AT1 receptor mRNA, with a maximum of 33.3% at 12 hours. There was a correlative decrease in AT1 receptor density.

Conclusions—This novel observation of estrogen-induced downregulation of AT1 receptor expression could explain the association of estrogen deficiency with hypertension and atherosclerosis, because activation of the AT1 receptor plays a key role in the regulation of blood pressure, fluid homeostasis, and vascular cell growth.

The low incidence of vascular diseases in premenopausal women and the rapid increase of the risk of cardiovascular events after menopause as well as the beneficial effects of estrogen replacement therapy on cardiac and vascular morbidity have suggested a important role of estrogens in the pathogenesis of atherosclerosis.123 In addition to its effects on classic cardiovascular risk factors, eg, in the sense of a decrease of cholesterol plasma levels,45 estrogen has been recognized to directly influence vascular as well as myocardial cells. Indeed, VSMCs, myocytes, and cardiac fibroblasts have been shown to contain functional estrogen receptors.678 Moreover, there is increasing evidence that estrogen interferes with the RAS. The production of angiotensinogen is enhanced, whereas ACE levels are decreased, by estrogens.9 According to a recent report, plasma renin levels are also reduced during estrogen replacement therapy, but other reports suggested either an increase or no change of plasma renin levels on estrogen treatment.10111213 One of the major components of the RAS is the AT1 receptor, which mediates most biological effects of Ang II, such as vasoconstriction, aldosterone release, sodium and water retention, and cellular growth.14 The expression level of the AT1 receptor is subject to regulation and governs the activity of the entire RAS through upregulation or downregulation. The AT1 receptor is regulated by, eg, lipoproteins, growth factors, and Ang II in vitro as well as in vivo, suggesting the important role of this receptor in the development of atherosclerosis.15161718 To explore a potential involvement of AT1 receptor regulation in the estrogen-induced modulation of cardiovascular diseases, we investigated the effects of estrogen deficiency on vascular AT1 receptor expression in ovariectomized rats and the direct effect of estradiol on AT1 receptor expression in VSMCs.

Functional Experiments

Rats were killed by decapitation 4 weeks after operation. Then the chest was rapidly opened, and the descending thoracic aorta was removed. The aorta was placed in chilled Krebs-Henseleit buffer and cleaned of excessive adventitial tissue. Eight 2- to 5-mm ring segments of thoracic aorta were suspended in individual organ chambers.18 When a stable baseline tone was established, potassium chloride, norepinephrine, endothelin, and Ang II were added at the concentrations indicated, interrupted by washout periods.

mRNA Isolation, Northern Analysis, Quantitative PCR

After the indicated treatments, cells or isolated aortas were lysed with RNA-clean (AGS) and processed according to the manufacturer’s protocol to obtain total cellular RNA. Aliquots (10 μg) were electrophoresed through formaldehyde agarose gels, transferred onto Hybond N membranes, and then hybridized for 15 hours at 42°C with a random-primed, [32P]dCTP-labeled rat AT1 receptor cDNA probe as described previously in detail.17 For quantitative PCR, isolated RNA was analyzed with a deletion-mutated AT1 receptor mRNA as internal standard. PCR was performed under the same conditions and with use of the same specific primers as described previously.19

Radioligand Binding Assays

VSMCs and aortic tissue were homogenized and membranes were isolated as described elsewhere.18 Ang II receptors were investigated in saturation experiments using 125I-labeled Ang II as radiolabeled ligand. The AT1 receptor antagonist DUP 753 (10 μmol/L) was used to determine nonspecific binding. The incubation was carried out at 24°C for 60 minutes. All experiments were performed in triplicate. The maximal density (Bmax) and apparent affinity (Kd) of binding sites were obtained from nonlinear regression analysis.

Statistical Analysis

Data are presented as mean±SEM. Statistical analysis was performed by the one-factor ANOVA test using the Scheffé procedure.

Renin plasma levels were measured to assess a possible compensatory modulation of the circulating RAS in response to the marked AT1 receptor overexpression. Indeed, renin concentrations were significantly lower in ovariectomized female rats (18.5±0.7 ng/mL) than in sham-operated female rats (23.3±1.8 ng/mL; P<0.05). Control experiments were conducted in which ovariectomized rats were substituted with exogenous estrogen. Figure 1E⇑ illustrates that aortic AT1 receptor mRNA was downregulated to control levels in ovariectomized female animals after estrogen treatment, suggesting a decisive role for estrogens in gene regulation of the vascular AT1 receptor. GAPDH mRNA remained unchanged (data not shown).

Most if not all vascular AT1 receptors are expressed in VSMCs, and AT1 receptor–mediated growth and vasoconstriction are realized predominantly through this cell type.14 To gain further mechanistic insight into the in vivo AT1 receptor regulation during estrogen deficiency, we investigated the effects of estradiol on VSMCs in culture. Control experiments showed that AT1 receptor and GAPDH mRNA levels remain stable over the experimental period of 24 hours (Figure 2A⇓). Estradiol (1 μmol/L) caused downregulation of AT1 receptor mRNA levels, with a maximal effect of 33.3±11% after a 12-hour incubation (Figure 2B⇓). Radioligand binding assays on cells treated for 12 hours with estradiol confirmed that, like the AT1 receptor mRNA, the AT1 receptor density was significantly downregulated from Bmax values of 1327.3±183.3 fmol/mg protein in controls to 776.9±49.5 fmol/mg protein in estradiol-treated cells (Figure 2C⇓). The receptor affinity was not significantly different (Kd=1.7 nmol/L [1.1 to 2.2 nmol/L] versus 2.7 nmol/L [1.2 to 4.2 nmol/L]).

Effect of estradiol on AT1 receptor expression in VSMCs. A and B, AT1 receptor steady-state levels. Time course of AT1 receptor (□) and GAPDH mRNA (○) in the presence of either vehicle (A) or 1 μmol/L estradiol (B) in VSMCs. Northern hybridizations were performed as described in “Methods.” Each point represents relative hybridization signal (mean±SEM) normalized to 0-hour treatment with vehicle (100%) from 5 separate experiments. C, AT1 receptor density. Confluent cells were exposed to either 1 μmol/L estradiol (○) or vehicle (□). Saturation binding assays using [125I]-labeled Ang II were performed on isolated membranes. AT1 receptor antagonist Dup753 (10 μmol/L) was used to define nonspecific binding. Each curve represents specific binding of radioligand (cpm radioligand bound minus cpm bound in the presence of 10 μmol/L losartan). Kd and Bmax values reported in the text were derived from nonlinear regression of specific bound versus free data. Each point represents binding data of 3 independent experiments ±SEM.

Discussion

Estrogen deficiency leads to upregulation of vascular AT1 receptor expression accompanied by an increased effect of Ang II on tension in isolated aortic rings. This is presumably based on a direct downregulating effect of estradiol on AT1 receptor gene expression in VSMCs.

VSMCs play a central role in the pathogenesis of atherosclerosis.20 Both estrogen and AT1 receptors are expressed in this cell type; therefore, estrogens and Ang II may influence the intracellular processes of VSMCs.614 The AT1 receptor decisively controls the events involved in VSMC growth and vasoconstriction.21 Because this receptor is subjected to homologous and heterologous regulation, its expression level governs the efficacy of the entire RAS. It is therefore conceivable that modulation of AT1 receptor expression may lead to acceleration of pathophysiological events involved in the development of vascular disease.

In addition, estrogens putatively participate in the atherosclerotic process suggested by the rapid increase of vascular events in women after menopause.123 Several mechanisms have been proposed that may initiate this atheroprotective effect of estrogens. Among others, beneficial influence on classic risk factors,45 scavenging of free radicals,22 and interference with the RAS have been reported. In this context, enhanced production of angiotensinogen1011 and reduced levels of ACE1323 have been observed, whereas the effect of estrogens on renin are the subject of ongoing controversy.101323

In ovariectomized rats, estrogen deficiency causes vascular overexpression of the AT1 receptor. In this short-term model, the blood pressure is not elevated significantly, probably because of the compensatory decrease of circulating renin levels. Our findings suggest that estrogen directly modulates AT1 receptor expression in VSMCs.

Recent reports showed that the AT1 receptor is overexpressed during conditions that are known to be associated with increased incidence of hypertension and atherosclerosis. LDL and salt cause a significant upregulation of AT1 receptor gene expression, leading to an enhanced biological efficacy of Ang II.171824 Hypercholesterolemia and increased salt load not only lead to an accelerated progression of cardiovascular diseases but also are associated with overexpression of AT1 receptors, indicating that this receptor regulation may indeed participate decisively in the development of hypertension and atherosclerosis. In analogy, the incidence of hypertension and atherosclerosis is increased in estrogen-deficient women,123 possibly because of increased vasoconstriction and cell growth via overexpressed AT1 receptors. Estrogen replacement therapy causes a decreased risk for cardiovascular diseases; AT1 receptors are consistently downregulated by estrogens. Therefore, it may be concluded that upregulation of AT1 receptors during estrogen deficiency and the premenopausal, physiological “downregulation” of AT1 receptors are involved in the estrogen-driven effects on onset and development of hypertension and atherosclerosis.

Selected Abbreviations and Acronyms

Ang II

=

angiotensin II

PCR

=

polymerase chain reaction

RAS

=

renin-angiotensin system

VSMC

=

vascular smooth muscle cell

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of Marc Wolff and Kerstin Löbbert is greatly appreciated.